PROJECT SUMMARY Platelets are critical for hemostasis, thrombosis and inflammatory responses. Billions of platelets circulate in mammalian blood to prevent blood loss in case of tissue injury. The lifespan of platelets is short (5-9 days in humans); as a consequence, several million platelets have to be produced every hour to maintain their physiological blood counts and to avoid the risk of bleeding. Platelets are generated in the bone marrow from megakaryocytes that, in turn, develop from hematopoietic stem and progenitor cells (HSPCs). Although important signaling pathways and transcription factors have been shown to play important roles in platelet biogenesis (thrombopoiesis), little is known about the epigenetic regulation of this process and the key epigenetic regulators involved. Because epigenetic regulators are essential components of regulatory networks that act collaboratively with transcription factors during cellular differentiation, lack of such knowledge significantly hinders the elucidation of the molecular networks controlling platelet generation and function. We have published significant work on the MLL3/4 (mixed lineage leukemia 3&4; KMT2C/KMT2D) histone methyltransferase (KMT) complex, including uncovering an important role for MLL4 in myeloid leukemia, a role for the MLL3/4 complex adaptor subunit PTIP (Pax interaction with transcription-activation domain protein-1) in lymphocyte class switch recombination and, recently, in maintaining normal and leukemic hematopoietic stem cell niches. For the latter studies, we have generated a mouse model of conditional inactivation of PTIP in HSPCs and consistently observed that PTIP deficiency led to thrombocytopenia. We recently generated a MK- specific mouse model and established that the observed reduction in platelets is an intrinsic effect of PTIP deletion in the MK lineage. Preliminary analysis performed on MLL4-deficient mice revealed mild, but significant, thrombocytopenia. Based on our preliminary data and published work, we hypothesize that PTIP is required for platelet generation by functioning together with MLL3 and MLL4 to direct a thrombopoiesis-specific gene expression program. The objective of this project is to determine the role of PTIP and its associated KMTs MLL3/4 in platelet generation. Results from the proposed studies are expected to fundamentally advance the fields of platelet biology by further defining the epigenetic mechanisms that regulate platelet generation. Finally, results from our work will likely contribute to the development of novel in vivo therapies aimed at enhancing platelet production or ex vivo expansion of platelets. Our long-term goal is to harness the reversibility of post-translation modifications of histones to promote human health. !